www.impactjournals.com/oncotarget/
Oncotarget, 2017, Vol. 8, (No. 37), pp: 62217-62230 Research Paper
CTCF promotes epithelial ovarian cancer metastasis by broadly controlling the expression of metastasis-associated genes Lintao Zhao1,4,*, Yang Yang1,*, Shigang Yin3,*, Tao Yang1, Jing Luo1, Rongkai Xie2, Haixia Long1, Lubin Jiang3 and Bo Zhu1 1
Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongqing, China
2
Department of Obstetrics and Gynecology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
3
Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
4
Institute of Cancer, PLA 324 Hospital, Chongqing, China
*
These authors have contributed equally to this work
Correspondence to: Haixia Long, email: [email protected] Lubin Jiang, email: [email protected] Bo Zhu, email: [email protected] Keywords: CTCF, metastasis, ovarian cancer, metastasis-associated genes, invasion Received: February 12, 2017 Accepted: April 17, 2017 Published: July 10, 2017 Copyright: Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT CCCTC-binding factor (CTCF) functions as both an oncogenic and a tumor suppressor, depending on the cancer type, through epigenetic regulation. Epigenetic regulation plays a key role in cancer metastasis. Our objective was to investigate whether CTCF plays a crucial role in epithelial ovarian cancer metastasis. First, we found that CTCF expression was increased in ovarian cancer tissues compared to non-tumor tissues. Increased expression of CTCF predicts poor prognosis of ovarian cancer patients. In addition, CTCF knockdown significantly inhibited the metastasis of ovarian cancer cells, although it had no effect on cell proliferation and tumor growth. More importantly, CTCF expression was higher in metastatic lesions compared to primary tumors from the same ovarian cancer patients. We also demonstrated that CTCF affects a number of metastasis-associated genes, including CTBP1, SERPINE1 and SRC. Additionally, our ChIP-seq results revealed that these genes have multiple CTCF-binding sites, findings that were further confirmed by ChIP-PCR. Our results suggest that CTCF could be a novel drug target to treat ovarian cancer by interfering with cancer cell metastasis.
to the development of novel therapeutic strategies and improve prognosis. Growing evidence indicates that tumor metastasis is mediated through the upregulation of metastasis-promoting genes [6], and the downregulation of metastasis suppressor genes [7]. In addition, besides changes in gene expression, epigenetic changes, including DNA methylation and histone modifications, also play an important role in tumor metastasis. Aberrant DNA methylation is a wellestablished molecular hallmark of metastatic lesions in various cancers [8, 9]. These methylation changes appear to regulate the expression of multiple genes that promote or suppress metastasis, including TIMP2 [10], CDH1 [11],
INTRODUCTION Ovarian cancer is one of the most common and lethal gynecological malignancies worldwide [1]. Over 90% of ovarian cancer cases are ovarian epithelial carcinomas [2]. Despite advances in treatment, the five-year survival rate of epithelial ovarian cancer remains at approximately 30% [3]. Moreover, greater than 60% of patients are diagnosed with advanced disease with intraperitoneal or distant metastasis [4]. The cure rate for ovarian cancer patients without or with metastasis is 88% and 18%, respectively [5]. Accordingly, elucidating the mechanisms underlying metastasis in epithelial ovarian cancer will contribute www.impactjournals.com/oncotarget
62217
Oncotarget
CXCL12 [12], and SERPINB5 [13], by regulating events, such as angiogenesis, epithelial-mesenchymal transition (EMT), migration, invasion and extravasation. Histone modifications, especially histone lysine methylation (HKM), are also associated with tumor metastasis. In particular, repressive histone modifications, such as H3K9 and H3K27 methylation, have been reported to inhibit genes, like SERPINB5 and DSC3, which could inhibit metastasis [14]. In ovarian cancer, a series of genes associated with metastasis have been shown to be downregulated by hypermethylation, including CDH1, CDKN2A and RARB [15]. However, the molecular mechanisms of these alterations are poorly understood. The CCCTC-binding factor (CTCF) is an 11 zinc finger transcription factor that binds more than 20,000 sites in the human genome [16], with the potential to mediate many epigenetic phenomena [17–20]. Previous studies have demonstrated that the expression pattern and role of CTCF vary in different tumor types. Sporadic mutations in CTCF or defective CTCF function have been observed in various cancer types [21–23], with CTCF functioning as a tumor suppressor. Disruption of promoter methylation, chromatin modifications and structural changes mediated by CTCF can lead to the dysregulation of cancer-associated genes, including RARRES1 [24], HOXA10 [25], TERT [26] and PTGS2 [27]. In contrast, some studies have suggested that CTCF may function as an oncogene in breast cancer [28] and acute lymphoblastic leukemia (ALL) [29]. However, the expression pattern of CTCF and its roles in ovarian cancer growth and metastasis remain unknown. Here, we report that CTCF expression was upregulated in ovarian cancer tissues and that increased CTCF expression was directly associated with poor prognosis of epithelial ovarian cancer patients. In addition, to the best of our knowledge, this study is the first to demonstrate that CTCF knockdown significantly suppressed ovarian cancer cell migration and invasion in vitro and tumor metastasis in vivo. Moreover, CTCF was also found to regulate a variety of metastasis-associated genes, including CTBP1, SERPINE1 and SRC. These findings suggest that CTCF may represent a novel therapeutic target for ovarian cancer metastasis intervention.
1A). The results of the IHC analysis also demonstrated significantly increased expression of CTCF in tumor specimens compared with the matched normal tissues (n=20, P=0.001; Figure 1B and 1C). In addition, we observed increased CTCF expression in epithelial ovarian cancer samples (n=57) compared with non-tumor samples (n=12) in a cohort of patients from a GEO database (GSE66957, P=0.009; Figure 1D). We additionally found that CTCF mRNA was upregulated in non-small-cell lung carcinoma (NSCLC, P=0.0061), gastric cancer (P=0.0339) and melanoma (P=0.0006), compared with the corresponding non-tumor tissues, by analyzing clinical data obtained from the GSE19804, GSE2685 and GSE8401 GEO databases, respectively (Supplementary Figure 1A-1C). To investigate the clinical significance of the CTCF expression changes in epithelial ovarian cancer, we analyzed the correlations between CTCF expression and tumor stage, tumor grade and 5-year overall survival rate using patient data obtained from the GEO databases GSE9891, GSE9891 and GSE13876, respectively. There were no significant differences in CTCF expression associated with different tumor stages (P>0.05; Figure 1E) or tumor grades (P>0.05; Figure 1F). Noteworthy, we found that CTCF expression was inversely correlated with the 5-year overall survival rate of ovarian cancer patients (P=0.027; Figure 1G). These data suggest that CTCF may play a role in the process of tumor progression.
Targeting CTCF in epithelial ovarian cancer cells has no effect on cell proliferation or tumor growth To further study the function of CTCF in epithelial ovarian cancer progression, we transfected 2 ovarian cancer cell lines (SKOV3 and A2780) with 2 different lentiviral shRNAs. Both shCTCF1 and shCTCF2 decreased CTCF mRNA expression by 60-70% in the 2 cell lines (P
Oncotarget, 2017, Vol. 8, (No. 37), pp: 62217-62230 Research Paper
CTCF promotes epithelial ovarian cancer metastasis by broadly controlling the expression of metastasis-associated genes Lintao Zhao1,4,*, Yang Yang1,*, Shigang Yin3,*, Tao Yang1, Jing Luo1, Rongkai Xie2, Haixia Long1, Lubin Jiang3 and Bo Zhu1 1
Institute of Cancer, Xinqiao Hospital, Third Military Medical University, Chongqing, China
2
Department of Obstetrics and Gynecology, Xinqiao Hospital, Third Military Medical University, Chongqing, China
3
Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
4
Institute of Cancer, PLA 324 Hospital, Chongqing, China
*
These authors have contributed equally to this work
Correspondence to: Haixia Long, email: [email protected] Lubin Jiang, email: [email protected] Bo Zhu, email: [email protected] Keywords: CTCF, metastasis, ovarian cancer, metastasis-associated genes, invasion Received: February 12, 2017 Accepted: April 17, 2017 Published: July 10, 2017 Copyright: Zhao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License 3.0 (CC BY 3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
ABSTRACT CCCTC-binding factor (CTCF) functions as both an oncogenic and a tumor suppressor, depending on the cancer type, through epigenetic regulation. Epigenetic regulation plays a key role in cancer metastasis. Our objective was to investigate whether CTCF plays a crucial role in epithelial ovarian cancer metastasis. First, we found that CTCF expression was increased in ovarian cancer tissues compared to non-tumor tissues. Increased expression of CTCF predicts poor prognosis of ovarian cancer patients. In addition, CTCF knockdown significantly inhibited the metastasis of ovarian cancer cells, although it had no effect on cell proliferation and tumor growth. More importantly, CTCF expression was higher in metastatic lesions compared to primary tumors from the same ovarian cancer patients. We also demonstrated that CTCF affects a number of metastasis-associated genes, including CTBP1, SERPINE1 and SRC. Additionally, our ChIP-seq results revealed that these genes have multiple CTCF-binding sites, findings that were further confirmed by ChIP-PCR. Our results suggest that CTCF could be a novel drug target to treat ovarian cancer by interfering with cancer cell metastasis.
to the development of novel therapeutic strategies and improve prognosis. Growing evidence indicates that tumor metastasis is mediated through the upregulation of metastasis-promoting genes [6], and the downregulation of metastasis suppressor genes [7]. In addition, besides changes in gene expression, epigenetic changes, including DNA methylation and histone modifications, also play an important role in tumor metastasis. Aberrant DNA methylation is a wellestablished molecular hallmark of metastatic lesions in various cancers [8, 9]. These methylation changes appear to regulate the expression of multiple genes that promote or suppress metastasis, including TIMP2 [10], CDH1 [11],
INTRODUCTION Ovarian cancer is one of the most common and lethal gynecological malignancies worldwide [1]. Over 90% of ovarian cancer cases are ovarian epithelial carcinomas [2]. Despite advances in treatment, the five-year survival rate of epithelial ovarian cancer remains at approximately 30% [3]. Moreover, greater than 60% of patients are diagnosed with advanced disease with intraperitoneal or distant metastasis [4]. The cure rate for ovarian cancer patients without or with metastasis is 88% and 18%, respectively [5]. Accordingly, elucidating the mechanisms underlying metastasis in epithelial ovarian cancer will contribute www.impactjournals.com/oncotarget
62217
Oncotarget
CXCL12 [12], and SERPINB5 [13], by regulating events, such as angiogenesis, epithelial-mesenchymal transition (EMT), migration, invasion and extravasation. Histone modifications, especially histone lysine methylation (HKM), are also associated with tumor metastasis. In particular, repressive histone modifications, such as H3K9 and H3K27 methylation, have been reported to inhibit genes, like SERPINB5 and DSC3, which could inhibit metastasis [14]. In ovarian cancer, a series of genes associated with metastasis have been shown to be downregulated by hypermethylation, including CDH1, CDKN2A and RARB [15]. However, the molecular mechanisms of these alterations are poorly understood. The CCCTC-binding factor (CTCF) is an 11 zinc finger transcription factor that binds more than 20,000 sites in the human genome [16], with the potential to mediate many epigenetic phenomena [17–20]. Previous studies have demonstrated that the expression pattern and role of CTCF vary in different tumor types. Sporadic mutations in CTCF or defective CTCF function have been observed in various cancer types [21–23], with CTCF functioning as a tumor suppressor. Disruption of promoter methylation, chromatin modifications and structural changes mediated by CTCF can lead to the dysregulation of cancer-associated genes, including RARRES1 [24], HOXA10 [25], TERT [26] and PTGS2 [27]. In contrast, some studies have suggested that CTCF may function as an oncogene in breast cancer [28] and acute lymphoblastic leukemia (ALL) [29]. However, the expression pattern of CTCF and its roles in ovarian cancer growth and metastasis remain unknown. Here, we report that CTCF expression was upregulated in ovarian cancer tissues and that increased CTCF expression was directly associated with poor prognosis of epithelial ovarian cancer patients. In addition, to the best of our knowledge, this study is the first to demonstrate that CTCF knockdown significantly suppressed ovarian cancer cell migration and invasion in vitro and tumor metastasis in vivo. Moreover, CTCF was also found to regulate a variety of metastasis-associated genes, including CTBP1, SERPINE1 and SRC. These findings suggest that CTCF may represent a novel therapeutic target for ovarian cancer metastasis intervention.
1A). The results of the IHC analysis also demonstrated significantly increased expression of CTCF in tumor specimens compared with the matched normal tissues (n=20, P=0.001; Figure 1B and 1C). In addition, we observed increased CTCF expression in epithelial ovarian cancer samples (n=57) compared with non-tumor samples (n=12) in a cohort of patients from a GEO database (GSE66957, P=0.009; Figure 1D). We additionally found that CTCF mRNA was upregulated in non-small-cell lung carcinoma (NSCLC, P=0.0061), gastric cancer (P=0.0339) and melanoma (P=0.0006), compared with the corresponding non-tumor tissues, by analyzing clinical data obtained from the GSE19804, GSE2685 and GSE8401 GEO databases, respectively (Supplementary Figure 1A-1C). To investigate the clinical significance of the CTCF expression changes in epithelial ovarian cancer, we analyzed the correlations between CTCF expression and tumor stage, tumor grade and 5-year overall survival rate using patient data obtained from the GEO databases GSE9891, GSE9891 and GSE13876, respectively. There were no significant differences in CTCF expression associated with different tumor stages (P>0.05; Figure 1E) or tumor grades (P>0.05; Figure 1F). Noteworthy, we found that CTCF expression was inversely correlated with the 5-year overall survival rate of ovarian cancer patients (P=0.027; Figure 1G). These data suggest that CTCF may play a role in the process of tumor progression.
Targeting CTCF in epithelial ovarian cancer cells has no effect on cell proliferation or tumor growth To further study the function of CTCF in epithelial ovarian cancer progression, we transfected 2 ovarian cancer cell lines (SKOV3 and A2780) with 2 different lentiviral shRNAs. Both shCTCF1 and shCTCF2 decreased CTCF mRNA expression by 60-70% in the 2 cell lines (P
